Adult dogs are capable of regulating calcium balance, with no adverse effects on health, when fed a high-calcium diet
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Downloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210 British Journal of Nutrition (2017), 117, 1235–1243 doi:10.1017/S0007114517001210 © The Authors 2017 Adult dogs are capable of regulating calcium balance, with no adverse effects on health, when fed a high-calcium diet Jonathan Stockman1, Phillip Watson1*, Matthew Gilham1, David Allaway1, Jujhar Atwal1, Richard Haydock1, Alison Colyer1, Helen Renfrew2 and Penelope J. Morris1 1 WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, Leicestershire LE14 4RT, UK 2 VetCT, St John’s Innovation Centre, Cowley Road, Milton, Cambridge CB4 0WS, UK (Submitted 6 February 2017 – Final revision received 20 April 2017 – Accepted 1 May 2017) Abstract Although the implications of long-term high Ca intakes have been well documented in growing dogs, the health consequences of Ca excess in adult dogs remain to be established. To evaluate the impact of feeding a diet containing 7·1 g/4184 kJ (1000 kcal) Ca for 40 weeks on Ca balance and health parameters in adult dogs, eighteen neutered adult Labrador Retrievers, (nine males and nine females) aged 2·5–7·4 years were randomised to one of two customised diets for 40 weeks. The diets were manufactured according to similar nutritional specifications, with the exception of Ca and P levels. The diets provided 1·7 and 7·1 g/4184 kJ (1000 kcal) (200 (SD 26) and 881 (SD 145) mg/kg body weight0·75 per d, respectively) Ca, respectively, with a Ca:P ratio of 1·6. Clinical examinations, ultrasound scans, radiographs, health parameters, metabolic effects and mineral balance were recorded at baseline and at 8-week intervals throughout the study. Dogs in both groups were healthy throughout the trial without evidence of urinary, renal or orthopaedic disease. In addition, there were no clinically relevant changes in any of the measures made in either group (all P > 0·05). The high-Ca diet resulted in a 3·3-fold increase in faecal Ca excretion (P < 0·001), whereas apparent Ca digestibility (%) and net Ca balance (g/d) did not significantly change from baseline or differ between the groups at any time point (both P > 0·05). Ca intakes of up to 7·1 g/4184 kJ (1000 kcal) are well tolerated over a period of 40 weeks, with no adverse effects that could be attributed to the diet or to a high mineral intake. Key words: Canine nutrition: Mineral balance: Digestibility: Calcium:phosphorus ratio Ca homoeostasis is tightly regulated in most vertebrate species concluded that the relative Ca faecal excretion is not altered as a through the interaction between several organs and glands such result of dietary Ca in adult dogs across a wide range of dietary as bone, gut, kidney, intestines and the parathyroid gland. Ca Ca intakes. This finding contrasts with expectations that relative concentrations in circulation are mediated through the actions Ca excretion would be very low when dogs are fed diets that of the parathyroid hormone (PTH) and calcitonin(1), although provide less Ca than the requirement, and that Ca excretion Ca homoeostasis is also maintained through adjustments in would approximate intake when Ca is provided above the intestinal absorption. Ca absorption in the mammalian small requirement. Instead, the results of the meta-analysis indicated intestine occurs via two mechanisms: a transcellular active a positive metabolic balance of Ca, where intake exceeded transport process, located largely in the duodenum and upper excretion by more than 11 % across a wide range of Ca intakes jejunum; and a paracellular, passive process that is present and was not affected by dog breed or the Ca source. A sub- throughout the intestine. When Ca intake is low, transcellular Ca sequent study(5) evaluated the impact of a diet containing Ca transport accounts for a substantial fraction of the absorbed Ca. at the National Research Council minimum requirement(6) of Conversely, when Ca intake is high, transcellular transport 0·5 g/4184 kJ (1000 kcal) in eleven adult female dogs (six Beagle accounts for a minor portion of the absorbed Ca, because of the and five Foxhound cross-breeds) over 6 months. This study short intestinal sojourn time and a down-regulation of Ca concluded that despite the low Ca intake throughout the period transporters dependent on 1,25(OH)2D(2). of feeding, there was seemingly no adaptation in Ca fractional Intestinal regulation of Ca absorption exists across many excretion leading to a negative Ca balance. In addition, an mammalian species and it has been widely accepted that increase in circulating PTH and vitamin D-metabolite con- a degree of regulation was also present in the dog(3). However, centrations as a result of the reduced Ca intake was not a recent meta-analysis of thirty-five Ca digestibility studies(4) detected, as might have been expected. Abbreviations: BW, body weight; iCa, ionised Ca; PTH, parathyroid hormone. * Corresponding author: Dr P. Watson, fax +44 1664 415440, email Phillip.Watson@effem.com
Downloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210
1236 J. Stockman et al.
This raises questions regarding the long-term adaptability of Table 1. Nutrient composition of the Control and Test diets analysed post
adult dogs to high dietary Ca. Lack of adaptation of faecal Ca manufacture
excretion in response to high intake may result in either a
Control Test
positive Ca balance or an increase in urinary Ca excretion;
however, previous data from growing dogs(7) showed that Ash (%) 5·2 10·7
urinary Ca excretion is generally low even when dogs were fed Fat (%) 12·5 11·9
Protein (%) 29·9 27·2
diets with high Ca content. Under conditions of long-term Ca Total dietary fibre (%) 5·8 5·1
excess, tissues may act as stores for Ca (e.g. bone) through Ca (g/4184 kJ (1000 kcal)) 1·7 7·1
changes in the degree of mineralisation. This has been P (g/4184 kJ (1000 kcal)) 1·1 4·5
Ca:P ratio 1·6 1·6
demonstrated during growth and development, with abnormal Na (g/4184 kJ (1000 kcal)) 1·2 1·1
skeletal development observed in large and giant-breed pups K (g/4184 kJ (1000 kcal)) 2·6 2·5
fed diets containing ≥5·5 g/4184 kJ (1000 kcal) Ca(3,7), but data Mg (mg/4184 kJ (1000 kcal)) 376 541
from adult dogs are not currently available. Fe (mg/4184 kJ (1000 kcal)) 42·2 80·0
Cu (mg/4184 kJ (1000 kcal)) 2·3 2·7
In humans, long-term dietary Ca excess of 2·4–4·6 g/d for up Mn (mg/4184 kJ (1000 kcal)) 18·5 17·7
to a year has been implicated to cause adverse effects, including Zn (mg/4184 kJ (1000 kcal)) 51 53
decreased dietary bioavailability of minerals such as Mg and I (mg/4184 kJ (1000 kcal)) 1·3 1·3
Se (µg/4184 kJ (1000 kcal)) 107 105
Zn, increased incidence of hypercalcaemia, hypercalciuria,
Chloride (g/4184 kJ (1000 kcal)) 2·3 2·4
urolithiasis, nephrolithiasis and non-reversible soft tissue calci- Vitamin A (µg/4184 kJ (µg/1000 kcal)) 1076 1362
fication(8). Dietary Reference Intakes for Ca and vitamin D have Vitamin E (mg/4184 kJ (mg/1000 kcal)) 89 112
been reviewed by the Institute of Medicine on the basis of Thiamin (mg/4184 kJ (1000 kcal)) 1·7 2·0
Riboflavin (mg/4184 kJ (1000 kcal)) 1·2 1·0
recent evidence related to both skeletal and extraskeletal Niacin/nicotinic acid (mg/4184 kJ (1000 kcal)) 13·3 11·8
outcomes(9). Former guidelines, set in 1997, established a Pyridoxine (mg/4184 kJ (1000 kcal)) 2·6 2·3
tolerable upper intake level (UL; the highest daily intake of the Pantothenic acid (mg/4184 kJ (1000 kcal)) 8·0 7·0
nutrient that is likely to pose no risk) for Ca at 2500 mg/d for all Folic acid (µg/4184 kJ (1000 kcal)) 483 358
Cyanocobalamin (µg/4184 kJ (1000 kcal)) 12 12
ages above 1 year, whereas the revised UL for Ca now range Choline (g/4184 kJ (1000 kcal)) 669 584
from 1000–3000 mg/d, depending on life-stage group(9). Vitamin D3 (µg/4184 kJ (µg/1000 kcal)) 5·2 5·0
Present guidelines for feeding adult dogs with Ca are based Base excess (MEq/kg)* 175 175
largely on data extrapolated from growth studies(6,10); conse- * Base excess = ([Ca2+] + [Mg2+] + [Na+] + [K+]) − ([PO4 3−] + [SO4 2−] + [Cl−]).
quently, the implications of long-term feeding of a high-Ca diet to
adult dogs remain unclear. Therefore, the current study aimed to
evaluate the impact of feeding a high-Ca diet for 40 weeks on Ca Royal Canin using similar ingredients and ratios to provide a
balance in adult dogs. Changes in renal function, related hor- similar nutrient composition, base excess and Ca:P ratio
mones and vitamin D metabolites, food intake, diet digestibility, (Table 1). To achieve this, both diets were composed of similar
and soft tissue and bone mineralisation were measured to support quantities of pork meal, animal fat, soya and fish oils, beet pulp,
the mineral balance data and to monitor the health of the dogs. cereal and flavourings, with the additional Ca and P in the test
diet provided through a combination of bone ash from protein
meal and supplemented calcium carbonate. The high-Ca diet
Methods
provided 7·1 g Ca/4184 kJ (1000 kcal), whereas the control diet
Dogs provided 1·7 g Ca/4184 kJ (1000 kcal); these inclusion levels
correspond to 710 and 170 % of the National Research Council
In all, eighteen neutered adult Labrador Retrievers (Labradors;
recommended allowance for the test and the control diets,
nine males and nine females) aged 2·5–7·4 years (median 3·2
respectively(6). The nutrient composition of both diets were
years) were selected for this study. All dogs underwent a pretrial
analysed at the Mars Petcare Europe Central Laboratory post
health assessment, including a physical examination and
production, and were compliant with both the American Asso-
haematological, biochemical and urine analysis, to ensure they
ciation of Feed Control Officials(10) and the National Research
were eligible for the study. Dogs were assigned to either Control
Council(6) recommended allowance values for adult dogs.
or Test (high-Ca) diet groups. The groups were stratified
according to sex, age, body weight (BW), familial affiliation (litter
mates) and energy requirements to maintain an ideal body Study design
condition score (/kg BW0·75). The Control group comprised eight
All dogs were fed the control diet during the first 9 weeks of the
dogs, whereas the Test group comprised ten dogs. The research
study. Baseline measures were obtained during weeks 8 and 9.
protocol was reviewed and approved by the WALTHAM©
Afterwards, the Test group was transitioned to a high-Ca diet for
Animal Welfare and Ethical Review Body and carried out under
a period of 40 weeks, whereas the Control group remained on
the authority of the Animals (Scientific Procedures) Act 1986.
the control diet. At the end of the test diet feeding, the Test
group was transitioned back to the control diet for an additional
Diets
5 weeks until the conclusion of the study.
Single batches of two dry extruded diets (the control diet and Dogs were housed according to their diet groups for the
the high-Ca test diet) were formulated and manufactured by duration of the study. During the days of urine and faecesDownloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210
High calcium intake in adult dogs 1237
sample collection, dogs were housed individually and moni- Urinalysis
tored 24 h/d to ensure all samples were collected and the dogs
A 5 ml freshly voided urine sample was collected from each dog
did not practice coprophagia. When individually housed, dogs
and analysed within 30 min for pH, glucose, protein, blood and
were still in the company of other dogs and were allowed 1 h/d
Hb (Siemens Multistix 10SG; Siemens AG). Following collection,
of supervised play interaction with their group. Food intake
specific gravity was assessed using a refractometer (Sinotech
and faecal quality were recorded daily. Fasted BW and body
RHCN-200ATC; Sinotech). The urine was then centrifuged at
condition score(11) were recorded weekly. Food provision
9391 g for 5 min and the sediment was separated and evaluated
was adjusted weekly for each dog to maintain an ideal body
under 20× magnification for evidence of crystals, erythrocytes,
condition score. All dogs had a full physical examination
leucocytes, bacteria or casts. Urine albumin was measured with a
performed by a researcher (J. S.) at each sampling occasion.
canine-specific ELISA (ab157685; Abcam PLC) and urinary crea-
Diet digestibility, mineral balance, glomerular filtration rate
tinine was measured using the Beckman Coulter creatinine assay
(GFR) and blood sampling for the health parameters outlined
(OSR6178; Olympus AU400 biochemistry analyser). The urinary
below were performed at baseline and at 8-week intervals
albumin:creatinine ratio was then calculated for each sample.
(8, 16, 24, 32 and 40 weeks) throughout the study. Radiography
and abdominal ultrasound scanning were performed at the
Glomerular filtration rate
beginning of the first test week and at subsequent 8-week
intervals. Dual-energy X-ray absorptiometry (DXA) was GFR was determined using the iohexol clearance method(12).
conducted at baseline and at weeks 24 and 40. Urine super In brief, 300 mg I/kg iohexol was administered over a 3-min
saturation was assessed at weeks 8, 16, 24, 32, 40 and 48. period, via a cephalic catheter, followed by a heparinised
saline flush. The completion of the injection represented time-
zero. Blood samples of 2 ml volume were collected from the
Tests and measures – complete blood count (haematology),
cephalic vein into gel-activated serum clot tubes at 2, 3 and
biochemistry and ionised calcium
4 h post iohexol infusion. Blood samples were then centrifuged at
A total of 1 ml of blood was drawn via the jugular veins on each 1999 g for 10 min, with the resulting serum frozen at –80°C until
sampling occasion. The blood was divided to three tubes analysis. Iohexol concentration in the serum of each dog at each
(EDTA–2× heparin) for immediate analysis. Complete blood time point was analysed using High-performance capillary electro-
counts were conducted using 0·2 ml of whole blood in EDTA phoresis (undertaken by the Department of Comparative and
using a haematology analyser (Mythic 18; Orphée SA). Plasma Biomedical Sciences, The Royal Veterinary College, London).
was separated from 0·5 ml of heparinised blood via cen- Clearance (in ml/kg per min) was calculated using the slope
trifugation at 1999 g for 10 min at 4°C before analysis for bio- of the concentration gradient over the three time points.
chemical variables (Olympus AU400 Biochemistry Analyzer;
Beckman Coulter Inc.). Ionised Ca (iCa), blood pH and blood Imaging
gas analysis was conducted using 0·3 ml of heparinised whole
Ventrodorsal and right lateral recumbent radiographs (AGFA
blood (Stat Profile Prime Critical Care Analyser; Woodley
CR 30-X, exposure 46–64 kV, 2·36–4·73 mAs; AGFA) and
Equipment Company Ltd).
ultrasound scans (GE Logiq-E with microconvex probe (8 C),
frequency range of 4·0–10·0 MHz) were acquired at each time
Parathyroid hormone, vitamin D metabolites, serum point. Examinations were performed in right followed by left
cross-laps (C-terminal telopeptide) and bone alkaline lateral recumbency with the dogs under sedation (induced
phosphatase through administration of 0·25 mg/kg Butorphanol, 10 µg/kg
Medetomidine and 0·2 mg/kg Midazolam). All scans and inter-
A 7 ml blood sample was divided between two gel-activated
pretation of the radiographs were carried out by a board-
clot tubes and allowed to clot for a minimum of 1 h before
certified veterinary radiologist (H. R.). DXA (EncoreTM 2006,
centrifugation for 10 min at 1999 g at room temperature. The
10.50 software; GE Lunar Prodigy Advance) was also performed
resultant serum samples were stored at –80°C before being
to determine bone mineral density (BMD; g/cm2) and bone
shipped on dry ice to external laboratories. Analysis of serum
mineral content (BMC; g). The dogs underwent a short sedation
PTH, vitamin D metabolites, serum cross-laps and bone alkaline
(as described above) for the completion of this procedure and
phosphatase (BAP) were conducted at Alomed, Radolfzell,
analysis was carried out in the supine position.
Germany. PTH was analysed using a direct luminometric
sandwich-immunoassay, which has been internally validated
Mineral balance and apparent digestibility
in dogs. 25-(OH)-vitamin D2 and 25-(OH)-vitamin D3 were
analysed by HPLC and 1,25-(OH)2-vitamin D3 was determined Faeces and urine were collected over 5 consecutive days at
by RIA. The bone formation marker BAP was analysed using a each time point. Urine was collected via a free-catch method. In
canine-specific enzyme-immunoassay (MicroVue Bone Health the case of urine spillage or a missed sample collection, urine
BAP EIA Kit; Quidel); the bone resorption marker serum volumes were estimated according to the urination duration and
cross-laps, which recognises the cross-linked carboxyterminal size of the puddle/carpet stain. Urine samples were analysed
telopeptides of type I collagen (CTx) concentrations in the for Ca, Mg (flame photometry) and P (spectral photometry)
serum, was analysed using a canine-specific ELISA kit (CTX-1 content, with these data used to determine the urinary fractional
ELISA; IDS). excretion of these minerals.Downloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210
1238 J. Stockman et al.
Faeces were stored frozen at –20°C in a sealed container until study in week 27 because of the diagnosis of a hepatic mass on
processing. Each pooled 5 d faecal collection was weighed and abdominal ultrasound scanning. The dog underwent a sub-
then freeze-dried (One VirTis BenchTop BTP9ES Freeze Dryer; sequent laparotomy and a partial hepatectomy, which revealed
Biopharma Process Systems). Once dried, the faeces were a benign hyperplastic mass. All other dogs were assessed as
weighed again and manually homogenised in a mortar and healthy and free of any clinical signs of adverse dietary effects
pestle. Any foreign bodies, such as rocks and plant material, were during the entire duration of the study. BW (ControlBaseline 27·3
removed and weighed separately. A 50 g aliquot of the homo- (95 % CI 24·7, 29·9) kg, ControlEnd 27·0 (95 % CI 24·5, 29·6) kg;
genate was sent for laboratory analysis, which included fat, TestBaseline 27·2 (95 % CI 25·0, 29·5) kg, TestEnd 27·2 (95 % CI
protein, DM, organic matter, fibre, ash, Ca, P, Zn and Mg analysis. 24·9, 29·5) kg; P = 1·000) and energetic intake (ControlBaseline
Apparent protein, fat, DM and mineral digestibility were calcu- 418 (95 % CI 347, 490) kJ/kg0·75, ControlEnd 397 (95 % CI
lated as the difference between the dietary intake and faecal 331, 469) kJ/kg0·75; TestBaseline 448 (95 % CI 385, 510) kJ/kg0·75,
content over the 5 collection days and expressed as percentage TestEnd 452 (95 % CI 385, 515) kJ/kg0·75; P = 1·000) were
of the intake. The total mineral balance (g) was also calculated not significantly different between the groups at any time.
for Ca, P and Mg as intake − (faecal excretion + urinary excretion). Following the baseline period, Ca intake was 200 (SD 26) mg/kg
BW0·75 per d for the Control group and 881 (SD 145) mg/kg
Urine mineral content and relative super saturation BW0·75 per d in the Test group.
All of the health-related measures (e.g. haematology, serum
All passed urine was collected over a 5 d period. The method
biochemistry) remained within normal ranges throughout
for urine relative super saturation (RSS) analysis has been pre-
the study and none showed consistent significant differences
viously described(13). Samples were analysed for oxalate,
between the groups (P > 0·05). iCa concentrations were not
citrate, pyrophosphate, K, Ca, Na, ammonium, chloride, sul-
different between groups before the intervention (Control 1·21
phate and phosphate via HPLC. The concentrations of analytes
(95 % CI 1·18, 1·25) mmol/l; Test 1·23 (95 % CI 1·20, 1·26) mmol/l;
were then used by SUPERSAT software (Institute of Urology
P = 0·995). Although there was a tendency for iCa concentrations
and Nephrology, University College) to calculate the RSS
to be lower in the Test group compared with the Control group
(activity product/solubility product) for struvite and Ca oxalate.
at week 8 (Control 1·27 (95 % CI 1·21, 1·33) mmol/l; Test 1·20
This analysis is carried out at the Royal Canin SAS European
(95 % CI 1·17, 1·23) mmol/l; P = 0·065), no significant differences
Regional Laboratory in Aimargues, France.
over time (P > 0·05) or between groups (P > 0·05) were apparent
throughout the study.
Statistical analysis
Power analyses by simulation were performed for the primary
Imaging
response variable (the percentage difference in intake and
excreted Ca levels (mg/kg0·75). Data from a previous study(5) Of the seventeen dogs that completed the study, one dog was
evaluating faecal Ca excretion were used to estimate the radiographed on the three initial occasions, but only underwent
between-dog variability to enable simulation of data sets. Using ultrasonographic examinations for the subsequent three time
this methodology, it was found that ten dogs were needed in the points because of concerns over response to sedation. No
Test group when using eight dogs in the Control group to detect evidence of urolithiasis or soft tissue mineralisation was detec-
a 6 % reduction, from a baseline of 12 %, in the percentage dif- ted on any occasion using either modality. Ultrasound scans
ference in percentage retention of Ca with at least 80 % power. highlighted mild pyelectasia in eleven of the dogs (seven from
Data are presented as means with 95 % CI. The data were the Control group, four from the Test group) on a single
analysed using linear mixed effects models with each measure as occasion during the study; however, this finding was transient
the response. Fixed effects of diet, sampling occasion and their and was not evident in follow-up scans.
interaction, and a random effect of dog, to account for the repeat
measurements, were examined. Three sets of hypothesis tests
Renal and urinary health
were performed: first, the difference between groups at each of
the six sampling occasions; second, the change from baseline of Serum creatinine and urea were not significantly different
each time point within each diet group; and finally, the difference between the groups at any time point or over time, with the
in the change from baseline between diet groups. All analyses exception of week 40 when both were significantly higher in
were performed using R version 3.2.0 with the lme4 and mult- the Control group (Table 2; P = 0·005). GFR calculated using the
comp libraries, and the hypothesis tests were corrected for iohexol clearance method (Fig. 1) was reduced compared with
multiplicity to control the family-wise error rate to a level of 5 %. baseline in both groups until 32 weeks (P < 0·05), with no
differences apparent between the two groups at any time point
(P > 0·05). Urine-specific gravity and the urinary albumin:
Results creatinine ratio did not change during the study or show
significant differences between the groups on any occasion. RSS
General health data
for both calcium oxalate and for magnesium ammonium
Of the eighteen Labrador Retrievers initially included, seven- triphosphate (struvite) did not show significant differences in
teen completed the full 56 weeks of the study. One female the between groups change from baseline (Table 2). There was
(aged 8·2 years) from the Test diet group was removed from the no evidence of urinary tract disease in any dog during the study.Downloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210
High calcium intake in adult dogs 1239
Table 2. Renal and urinary health measures*
(Mean values and 95 % confidence intervals)
Control Test
Weeks Mean 95 % CI Mean 95 % CI P
Serum creatinine (µmol/l)
Baseline 112·4 104·0, 121·5 112·2 104·7, 120·3 1·000
8 114·5 105·9, 123·7 108·8 101·5, 116·7 0·883
16 116·1 107·4, 125·5 115·3 107·6, 123·6 1·000
24 116·6 107·9, 126·0 112·5 104·9, 120·6 0·987
32 122·9 113·7, 132·9 112·1 104·4, 120·4 0·193
40 131·1 121·3, 141·6 113·3 105·5, 121·6 0·005
Serum urea (mmol/l)
Baseline 5·8 5·1, 6·7 6·5 5·7, 7·3 0·800
8 5·5 4·8, 6·4 5·0 4·4, 5·7 0·787
16 6·2 5·4, 7·2 5·7 5·0, 6·4 0·851
24 6·3 5·5, 7·3 5·8 5·1, 6·5 0·902
32 6·2 5·4, 7·2 5·7 5·0, 6·4 0·859
40 6·5 5·7, 7·5 5·3 4·7, 6·0 0·058
Urine-specific gravity
Baseline 1·023 1·010, 1·035 1·026 1·015, 1·037 1·000
8 1·018 1·005, 1·030 1·020 1·008, 1·031 1·000
16 1·022 1·010, 1·035 1·020 1·009, 1·031 1·000
24 1·024 1·011, 1·036 1·015 1·004, 1·026 0·844
32 1·017 1·004, 1·029 1·010 0·999, 1·022 0·977
40 1·019 1·006, 1·031 1·018 1·006, 1·029 1·000
Urine RSS struvite
8 2·50 0·44, 14·10 0·15 0·03, 0·73 0·015
16 1·83 0·32, 10·31 0·18 0·04, 0·84 0·078
24 1·25 0·22, 7·07 0·30 0·06, 1·41 0·651
32 1·30 0·23, 7·31 0·10 0·02, 0·49 0·039
40 0·49 0·09, 2·74 0·12 0·02, 0·58 0·672
48 0·21 0·04, 1·17 0·34 0·07, 1·65 1·000
Urine RSS calcium oxalate
8 2·58 1·03, 6·43 2·07 0·92, 4·70 1·000
16 3·61 1·45, 8·99 2·04 0·90, 4·62 0·909
24 2·18 0·87, 5·43 1·71 0·75, 3·86 1·000
32 2·59 1·04, 6·46 1·54 0·67, 3·56 0·951
40 2·80 1·12, 6·98 3·28 1·42, 7·55 1·000
48 2·36 0·95, 5·89 3·16 1·37, 7·29 0·999
RSS, relative super saturation.
* P values represent comparison between groups at the given time point.
2.50 different between the Control and the Test groups in any of the
measurements (Table 3). Similarly, bone formation (BAP; P > 0·05)
and bone resorption markers (serum CTx; P > 0·05) as well as the
2.25
measured hormones and vitamin D metabolites (PTH, fibroblast
GFR (ml/kg per min)
growth factor 23 (FGF23), calcidiol and calcitriol) were not signi-
ficantly different between the groups at any time point (Table 3).
2.00
Mineral balance and apparent digestibility
1.75
There was no difference in faecal Ca excretion between the
groups at baseline (P = 0·913). Ingestion of the Test diet resulted
1.50 in a 3·3-fold elevation in faecal Ca excretion for the remainder
Baseline 8 16 24 32 40
of the study (P < 0·001), with no change apparent in the Control
Weeks
group (Fig. 2(a)). Urinary Ca excretion was increased in the Test
Fig. 1. Glomerular filtration rate (GFR) measured throughout the study in both group at weeks 8, 16, 24 and 40 (Fig. 2(a); P < 0·05), although
the Control ( ) and the Test ( ) groups. Values are means and 95 % CI. urinary Ca losses remained considerably lower than those
measured in the faeces. Consequently, total Ca balance was not
significantly different from 0 at any time point in either group
Bone health and related hormones
(Fig. 2(b)). The estimated difference between groups in the
DXA measurements for bone mineralisation including BMD change in Ca balance over the duration of the study was 0·85
(g/cm2; P > 0·05) and BMC (g; P > 0·05) were not significantly (95 % CI –1·67, 3·36) g (P = 0·983). A linear regression of dietaryDownloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210
1240 J. Stockman et al.
Table 3. Bone measures and related hormones*
(Mean values and 95 % confidence intervals)
Control Test
Weeks Mean 95 % CI Mean 95 % CI P
DXA BMC (g)
Baseline 965 838, 1110 939 828, 1065 0·999
24 937 815, 1078 927 817, 1050 1·000
40 961 836, 1106 956 843, 1084 1·000
DXA BMD (g/cm2)
Baseline 0·99 0·94, 1·05 0·99 0·94, 1·04 1·000
24 1·00 0·94, 1·06 1·00 0·95, 1·06 1·000
40 1·00 0·94, 1·06 1·00 0·95, 1·06 1·000
Bone alkaline phosphatase (UL)
Baseline 8·9 6·9, 11·6 9·3 7·3, 11·7 1·000
8 11·0 8·5, 14·3 9·5 7·5, 12·0 0·942
16 8·2 6·3, 10·7 9·6 7·6, 12·2 0·919
24 8·3 6·4, 10·8 8·5 6·7, 10·8 1·000
32 9·8 7·6, 12·8 9·4 7·3, 12·0 1·000
40 9·9 7·6, 12·9 9·1 7·1, 11·6 0·999
Serum cross-laps (ng/ml)
Baseline 0·83 0·41, 1·67 0·48 0·27, 0·86 0·711
8 0·76 0·38, 1·54 0·45 0·25, 0·81 0·758
16 0·60 0·30, 1·21 0·38 0·21, 0·68 0·880
24 0·72 0·36, 1·45 0·53 0·30, 0·96 0·992
32 0·37 0·19, 0·75 0·42 0·23, 0·75 1·000
40 0·41 0·20, 0·82 0·24 0·13, 0·43 0·748
PTH (ng/ml)
Baseline 14·2 9·4, 21·4 18·3 12·9, 26·0 0·886
8 15·4 10·4, 22·7 15·6 11·0, 22·2 1·000
16 17·8 12·0, 26·2 18·6 13·1, 26·4 1·000
24 17·3 11·7, 25·5 20·8 14·7, 29·5 0·980
32 15·3 10·4, 22·7 22·1 15·4, 31·8 0·487
40 18·6 12·6, 27·4 20·5 14·2, 29·5 1·000
FGF23 (pg/ml)
Baseline 377 305, 466 486 402, 587 0·169
8 385 311, 475 436 355, 536 0·944
16 390 316, 482 452 374, 546 0·827
24 362 293, 447 432 356, 524 0·661
32 377 305, 466 415 343, 505 0·988
40 401 325, 496 434 357, 527 0·998
Calcidiol (µg/l)
Baseline 43·8 37·8, 50·9 43·2 37·8, 49·4 1·000
8 39·5 34·0, 45·8 36·6 32·1, 41·9 0·979
16 39·5 34·0, 45·8 35·0 30·6, 40·0 0·695
24 37·4 32·2, 43·4 33·2 29·0, 37·9 0·709
32 40·7 35·0, 47·3 34·2 29·8, 39·2 0·225
40 38·4 33·1, 44·6 35·6 31·1, 40·8 0·980
Calcitriol (µg/l)
Baseline 16·7 12·9, 21·6 15·7 12·5, 19·8 1·000
8 14·6 11·2, 18·9 16·0 12·5, 20·3 0·999
16 15·9 12·3, 20·6 16·8 13·2, 21·3 1·000
24 15·0 11·6, 19·5 17·6 13·8, 22·4 0·931
32 13·0 10·0, 16·8 16·7 13·1, 21·2 0·457
40 13·2 10·2, 17·1 16·0 12·6, 20·4 0·774
DXA, dual-energy X-ray absorptiometry; BMC, bone mineral content; BMD, bone mineral density; UL, upper intake level; PTH, parathyroid hormone;
FGF23, fibroblast growth factor 23.
* P values represent comparison between groups at the given time point.
Ca intake v. faecal Ca excretion throughout the study high- on week 40 (P = 0·003), no further differences between groups
lighted that the 95 % confidence regions for both diet groups were apparent (all P > 0·05). There were no significant differ-
surround the y–x line, supporting the view that there was no ences between the groups in P or Mg digestibility over the
difference in the amount of Ca retained n either the Control or duration of the study. During the 8 weeks following transition
the Test diets (Fig. 3). to the test diet, Zn digestibility was reduced compared with
Apparent Ca digestibility was not different between groups at baseline (Baseline 3·0 (95 % CI –14·3, 20·3) %; week 8 –23·3
the end of baseline-diet feeding (Control 19·3 (95 % CI –1·1, (95 % CI –40·6, −6·0); P = 0·026) and was significantly lower
39·7) %; Test 5·0 (95 % CI –2·1, 22·1) %; P = 0·782). With the than that in the Control group at the same time point
exception of Ca digestibility being elevated in the Control group (P = 0·009). This response was transient, with no differencesDownloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210
High calcium intake in adult dogs 1241
(a) 12.5 for a transient fall in protein digestibility during the initial
8 weeks of test diet feeding (Control 85·8 (95 % CI 83·0, 88·6) %;
10 Test 80·6 (95 % CI 78·1, 83·1) %; P = 0·059), there was no
significant change in energy, protein or fat digestibility or any
Ca excretion (g/d)
7.5 differences between groups throughout the remainder of the
Faecal
study (P > 0·05). From week 8, there was a significant reduction
5
in DM digestibility apparent in the high-Ca diet group (78·2
(95 % CI 76·1, 80·4) %) when compared with the Control group
2.5
(88·2 (95 % CI 85·8, 90·6) %; P < 0·001). This response remained
until the end of the trial.
Urinary
0.015
0.005
Baseline 8 16 24 32 40 Discussion
Weeks
The aim of this study was to evaluate the impact of feeding a
(b) high-Ca diet on markers of health and Ca balance in adult dogs.
2 Although concerns have been raised regarding the long-term
feeding of Ca excess in dogs(4,5,14), the current study showed
that both the control and the high-Ca diets were well tolerated,
1
Ca balance (g/d)
and no dogs displayed gastrointestinal signs, reduced food
intake, failure to maintain ideal body condition or any adverse
0 effects that could be attributed to the diet or to a high mineral
intake. In addition, there were no consistent differences
–1 between the Test and the Control groups in any measured
haematological and biochemical parameters, urinalysis, or GFR,
which could indicate clinical or sub clinical negative effects as a
–2
Baseline 8 16 24 32 40 result of a high Ca intake. It should be noted that one of the
Weeks dogs in the Test group was removed from the study in week 27
because of the ultrasonographic diagnosis of a hepatic mass.
Fig. 2. Faecal and urinary Ca excretion (a) and Ca balance (b) throughout the
study in both the Control ( ) and the Test ( ) groups. Values are Histopathology of the mass following excision revealed that the
means and 95 % CI. mass was benign and without evidence of mineralisation or any
other pathologic finding that could suggest a connection to
high Ca intake.
Despite marked differences in dietary Ca intake, iCa
1 remained stable throughout the study and did not differ
Ca excretion (g/MBW)
between groups at any time point. Regulation of circulating Ca
concentrations involves a complex process, reliant on the
0.32 integrated actions of PTH, vitamin D metabolites, calcitonin and
iCa itself on Ca-sensing receptors expressed in the parathyroid
gland and the renal tubules of the kidney(1). Increased dietary
0.1 Ca intake results in a reduction in the synthesis and release of
PTH, which exerts both direct and indirect effects on the
intestine, kidney and bone. Reduced circulating PTH controls
0.03 the synthesis of calcitriol (1,25 (OH)2-vitamin D) by suppressing
0.1 0.18 0.32 0.56 1 renal 1-α-hydroxylation of calcidiol (25(OH)-vitamin D) derived
Ca intake (g/MBW) from the circulation. Through this response, reduced circulating
Fig. 3. Linear regression between dietary Ca intake and faecal Ca excretion calcitriol concentrations will limit Ca absorption from the
(g/kg body weight (BW)0·75; metabolic body weight (MBW)). The 95 % confidence intestine, resulting in an increase in faecal Ca excretion. In
regions for both diet groups surround the y–x ( ), supporting the view that addition, reductions in PTH and calcitriol, in response to high
there was no difference in the amount of Ca retained in either the Control ( )
or the Test ( ) diets.
Ca intakes, will suppress osteoclastic bone resorption and
reduce renal Ca reabsorption. Whereas there was a significant
compared with the baseline or with the Control group during reduction in serum calcitriol concentrations in the Test diet
the remainder of the study (P > 0·05). group over the course of the study, at no point was this
No differences in energy (Control 86·4 (95 % CI 83·6, 89·2) %; response different to the corresponding time point in the
Test 83·8 (95 % CI 81·5, 86·2) %; P = 0·434), protein (Control 85·7 Control group, suggesting some seasonal variation in this
(95 % CI 82·7, 88·6) %; Test 84·9 (95 % CI 82·4, 87·4) %; metabolite of vitamin D. In addition, a high dietary Ca intake
P = 1·000) or fat (Control 97·5 (95 % CI 96·7, 98·3) %; Test 97·8 failed to significantly alter serum PTH concentrations. The lack
(95 % CI 97·1, 98·5) %; P = 1·000) digestibility were apparent of a clear diet effect on these measures is surprising, but it may
between groups during baseline feeding. Despite a tendency be explained by the nutritional adequacy of both diets (e.g. notDownloaded from https://www.cambridge.org/core. IP address: 46.4.80.155, on 02 Feb 2021 at 14:25:19, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0007114517001210 1242 J. Stockman et al. sufficiently different to elicit a response) and the similar Ca:P demonstrate an altered faecal excretion of these minerals or a ratio (1·6:1) of the control and test diets, or it is possible that decrease in fat or protein digestibility in the Test group, as might some adaptation had already occurred during the first 8 weeks be expected. Similarly, there was no difference in energy intake that was missed by the first sample time point. required to maintain an ideal body condition between the Test A high dietary Ca intake resulted in increases in both faecal and the Control groups. This further supports the conclusion and urinary Ca excretion, with faecal excretion comprising a that Ca intake did not significant influence energy or macro- much greater proportion of overall losses. This observation is nutrient digestibility. A marked reduction in DM digestibility consistent with the view that a reduction in intestinal Ca was observed following transition to the test diet, with this absorption is a primary mechanism in the regulation of Ca response maintained for the duration of the study in this group; balance during exposure to excess dietary Ca in mammalian this response is most likely driven by the marked difference in species(1–3). It is worth noting that faecal excretion is comprised the ash content of the experimental diets (Control 5·2 %, Test of both unabsorbed dietary Ca, as well as a contribution from 10·7 %; Table 1). It was previously suggested that faecal Ca endogenous Ca losses. Further research may help identify excretion may be negatively associated with dietary DM whether endogenous losses change in response to marked digestibility(18). As such, an increase in Ca absorption could changes in dietary Ca intake. Ca balance and apparent Ca result in a positive Ca balance in diets with very high digest- digestibility (expressed as a percentage of Ca intake) were not ibility, such as experimental or home-prepared diets. Com- significantly different between dogs fed the control or test diets. mercial dog diets contain raw ingredients and undergo As Ca balance was not significantly different from zero for either processing such as extrusion and cooking, which generally limit diet at any time point, the current results do not support the DM digestibility, and consequently lower Ca availability. This hypothesis that dogs are unable to sufficiently adjust Ca effect of Ca on diet digestibility may account for the difference intestinal absorption to account for an increased Ca intake(4). in the findings of the current study, compared with those pre- The results of the present study suggest that maintaining Ca sented in the meta-analysis by Mack et al.(4) where many of balance is a somewhat variable process as individual dogs had the diets evaluated were experimental diets. It is also worth positive or negative balances at different sampling occasions noting that the studies included in this meta-analysis involved throughout the trial regardless of diet; this may also represent a relatively short-term exposure to the experimental diets, and phased regulation of Ca balance, where small quantities of Ca that much of the data were drawn from studies feeding Ca accumulate up to a threshold that triggers excretion to maintain below minimum requirements, as opposed to intakes compar- balance over time. As these effects were neither consistent able with those in the present study. within diet, or maintained within particular animals over time, There are a number of factors that must be considered when overall Ca balance was determined as neutral in both groups, translating the results of this study into canine diet formulations. which may hide a more individualised response. It is also worth First, dietary factors, including dietary content of phytate, noting that despite a 2·1-fold increase in urinary Ca excretion, oxalate, fat, the Ca source and the Ca:P ratio are thought to there was no concurrent increase in the RSS for Ca oxalate. influence Ca absorption(19); therefore, the diets in the present Therefore, it would appear that higher Ca intake does not study will not represent all possible diet formulations equate a high risk for Ca oxalate urolithiasis, at least in the range available in the market. The source of Ca in the current test diet of intakes studied here. was provided by a combination of pork meal and supple- Previous studies demonstrated that adult dogs exposed to a mented Ca carbonate. Ca derived from organic raw materials is low Ca intake (0·5 g/4184 kJ (1000 kcal)) for 6 months do not thought to be less bioavailable than Ca from other sources, but appear to adjust their fractional Ca excretion, leading to a there is evidence to suggest that organic and inorganic forms of negative Ca balance and a resulting mobilisation of bone Ca have similar bioavailability in many species(4,20). Finally, the minerals to maintain Ca homoeostasis(5). In contrast, the present present study was conducted in a single adult breed (Labrador study indicates that dogs are able to adequately regulate Ca Retriever). Previous studies in growing dogs(3,7) have docu- balance when diets contain levels above minimum require- mented that the effects of high-Ca diets vary in dogs of different ments. Given that several studies have highlighted abnormal breed sizes and that some implications of high mineral load, skeletal growth in large- and giant-breed puppies fed diets such as urolithiasis, are more common in certain breeds(21). containing ≥5·5 g/4184 kJ (1000 kcal) Ca(3,7), the present data Therefore, before generalised recommendations can be made, suggest a developmental adaptation enabling adult dogs to the impact of high Ca intakes should be investigated in regulate Ca uptake from the gastrointestinal tract, thus limiting other breeds. potential long-term health concerns when fed high-Ca diets. In conclusion, the current study demonstrates that Ca intakes This makes sense from an evolutionary perspective, supporting of up to 7·1 g/4184 kJ (1000 kcal) were well tolerated over a scavenging behaviours where dogs would have been likely to period of 40 weeks, with no adverse effects that could be rapidly consume large, infrequent meals high in Ca-rich bony attributed to the diet or to a high mineral intake. These data material, rather than Ca-deficient foods(15). provide evidence that adult dogs are able to tolerate higher Ca High dietary Ca has been suggested to cause chelation of fat intakes than puppies, and this will enable more precise and decrease absorption of dietary minerals such as P, Zn and recommendations for a safe Ca upper limit in adult dogs. In Mg, as demonstrated by studies in humans and rodents(13,16,17). addition, Ca balance was maintained throughout the study and The apparent digestibility and mineral balance results did was not significantly different from 0 in either group, at any reveal a transient reduction in Zn digestibility, but failed to time point. Under the present study conditions, there was no
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High calcium intake in adult dogs 1243
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